Process for the synthesis of pyrazolopyrimidines

ABSTRACT

The present invention provides a nucleoside comprising a pyrazolopyrimidine base and a process for producing the same. In particular, the processes of the present invention comprises using a halogenated pyrazolopyrimidine base and removing the halogen after the base is coupled to a sugar moiety. The presence of the halogen on the nucleoside base allows facile and economical production of a large quantity of nucleosides.

FIELD OF THE INVENTION

The present invention relates to a process for producingpyrazolopyrimidines. In particular, the present invention relates to aprocess for producing a PPG phosphoramidite.

BACKGROUND OF THE INVENTION

PPG phosphoramidite is often used in synthesis of nucleotides andoligonucleotides containing pyrazolopyrimidine base(s). Thesenucleotides and oligonucleotides are useful in a variety ofapplications, including as crosslinkable probes for use in therapeuticand diagnostic applications. See for example PCT publication No. WO90/14353, which is incorporated herein by reference in its entirety. Inaddition, oligonucleotides in which one or more purine residues havebeen replaced by pyrazolopyrimidines display enhanced duplex andtriplex-forming ability and display enhanced mismatch discriminationability. See, for example, Belousov et al., Nucleic Acids Res., 1998,26, 1324-1328 and U.S. Pat. Nos. 5,594,121 and 6,127,121, all of whichare incorporated herein by reference in their entirety. Furthermore, PPGcontaining oligonucleotides have been shown to reduce self-associationobserved with guanine-rich oligonucleotides and quenching observed whenguanine is adjacent to some fluorophore-containing oligonucleotideconjugates. See WO 00/142505.

Conventional syntheses of PPG phosphoramidite involve coupling anactivated sugar moiety with a pyrazolopyrimidine base to produce anucleoside containing pyrazolopyrimidine base. See, for example, Seelaet al., Helv. Chim. Acta, 1986, 69, 1602-1613. Further reactions arethen carried out to produce PPG phosphoramidite. Unfortunately,conventional processes for producing PPG phosphoramidite require anumerous chromatography separations including that of the nucleosideproduced from coupling an activated sugar moiety to a pyrazolopyrimidinebase. Typically, chromatography purification of a compound is notsuitable for a large scale (e.g., tens or hundreds of grams or more)production because a chromatography process is generally time consumingand labor intensive. Moreover, a large scale chromatography purificationprocess requires a correspondingly large amount of chromatographymaterial, e.g., silica gel and chromatography solvent(s), whichincreases the production cost of PPG phosphoramidite. Because thepyrazolopyrimidine base containing nucleoside is produced relativelyearly in the conventional synthesis of PPG phosphoramidite, purificationof this nucleoside by chromatography is one of the major hindrances in acost effective large scale PPG phosphoramidite synthesis.

Therefore, there is a need for a process which is amenable for a largescale production of PPG phosphoramidite.

SUMMARY OF THE INVENTION

One aspect of the present invention provides a PPG phosphoramiditecomprising a photolabile hydroxy protecting group, wherein saidphosphoramidite nucleoside is of the formula:

wherein

-   -   R¹ is selected from the group consisting of hydrogen and alkyl;    -   R² is selected from the group consisting of hydrogen, alkyl, and        an amine protecting group, or R¹ and R² together form an amine        protecting group;    -   each of Z¹, Z², Z⁴, and Z⁶ is independently selected from the        group consisting of hydrogen, halide, alkyl, —OR¹¹, wherein each        R¹¹ is independently selected from the group consisting of        hydrogen, alkyl, and a hydroxy protecting group or two R¹ groups        form a diol protecting group, or Z² and Z⁴ together with the        carbon atoms to which they are attached and C-3 carbon atom of        the carbohydrate ring form a five-to seven membered ring; and    -   one of Z³ or Z⁵ is —OR¹² and the other is —OR³, where R¹² is a        photolabile hydroxy protecting group and R¹³ is a        phosphoramidite.

Another aspect of the present invention provides a process for producinga nucleoside or nucleotide comprising a pyrazolopyrimidine base. Inparticular, the processes of the present invention comprise using ahalogenated pyrazolopyrimidine base and removing the halogen after thebase is coupled to a sugar moiety. The presence of a halogen on the baseallows the nucleoside to be purified by a non-chromatography method,preferably by recrystallization or precipitation. Thus, processes of thepresent invention allow economical production of a large quantity ofnucleosides, including PPG phosphoramidite.

Definitions

“Alkyl” means a linear or branched saturated monovalent hydrocarbonmoiety having from one to ten, preferably one to six, carbon atoms.Exemplary alkyl groups include, but are not limited to, methyl, ethyl,n-propyl, 2-propyl, tert-butyl, pentyl, and the like.

“Nucleoside” refers to a compound comprising a nucleoside base and asugar which are covalently attached.

“Nucleotide” refers to a compound comprising a nucleoside base, a sugarwhich are covalently attached to the nucleoside base, and a phosphorylgroup which is covalently attached to the sugar.

“Nucleoside base” refers to a non-carbohydrate derivative portion of thenucleoside or nucleotide, including both conventional bases such aspurine and pyrimidine bases and their modified versions such asdeazapyrimidine, pyrazolopyrimidines and the like.

“Sugar” refers to carbohydrate derivative portion of a nucleoside ornucleotide.

The terms “halo”, “halide” and “halogen” are used interchangeably hereinand refer to fluoro, chloro, bromo, or iodo, preferably chloro or iodo.

The term “photolabile hydroxy protecting group” refers to hydroxyprotecting groups that can be removed under photolytic conditions or acombination of acidic and photolytic conditions.

“Protecting group” refers to a moiety other than an alkyl groups thatwhen attached to a reactive group in a molecule masks, reduces orprevents that reactivity. Examples of protecting groups can be found inBeaucage and Iyer, Tetrahedron, 1992, 48, 2223-2311; T. W. Greene and P.G. M. Wuts, Protective Groups in Organic Synthesis, 3^(rd) edition, JohnWiley & Sons, New York, 1999; and Harrison and Harrison et al.,Compendium of Synthetic Organic Methods, Vols. 1-8 (John Wiley and Sons,1971-1996), which are incorporated herein by reference in theirentirety. Representative hydroxy protecting groups include acyl groups,benzyl and trityl ethers, tetrahydropyranyl ethers, trialkylsilyl ethersand allyl ethers. Representative amino protecting groups include,formyl, acetyl, trifluoroacetyl, benzyl, benzyloxycarbonyl (CBZ),tert-butoxycarbonyl (Boc), trimethyl silyl (TMS),2-trimethylsilyl-ethanesulfonyl (SES), trityl and substituted tritylgroups, allyloxycarbonyl, 9-fluorenylmethyloxycarbonyl (FMOC),nitro-veratryloxycarbonyl (NVOC), amidines, formamidines, and the like.

The terms “contacting,” “treating,” and “reacting” when referring to areaction between two or more reagents are used interchangeably hereinand refer to adding two or more reagents into a single reaction vessel,preferably in the presence of a solvent. It should be appreciated,however, the resulting reaction product can be produced directly from areaction between the added reagents or from an intermediate from one ormore of the added reagents which can be produced in the reactionmixture.

“Leaving group” has the meaning conventionally associated with it insynthetic organic chemistry, i.e., an atom or a group capable of beingdisplaced by a nucleophile and includes halo (such as chloro, bromo, andiodo), alkanesulfonyloxy, arenesulfonyloxy, alkylcarbonyloxy (e.g.,acetoxy), arylcarbonyloxy, mesyloxy, tosyloxy,trifluoromethanesulfonyloxy, aryloxy (e.g., 2,4-dinitrophenoxy),methoxy, N,O-dimethylhydroxylamino, pixyl and the like.

As used herein, the terms “those defined above” and “those definedherein” when referring to a variable incorporates by reference the broaddefinition of the variable as well as preferred, more preferred and mostpreferred definitions, if any.

DETAILED DESCRIPTION

One aspect of the present invention provides a PPG phosphoramiditecomprising a photolabile hydroxy protecting group. In particular, thephosphoramidite nucleoside is of the formula:

where R¹, R², Z¹, Z², Z³, Z⁴, Z⁵ and Z⁶ are those defined above.

In one particular embodiment of the present invention, the PPGphosphoramidite is of the formula:

where R¹, R², Z³ and Z⁵ are those defined above.

In another embodiment of the present invention, Z³ is —OR¹³ and Z⁵ is—OR¹², where R¹² and R¹³ are those defined above.

The photolabile hydroxy protecting group can be any conventionalphotolabile hydroxy protecting group that is used in nucleotidesynthesis. Such photolabile hydroxy protecting groups are well known toone skilled in the art and exemplified, for example, in U.S. Pat. Nos.5,889,165; 5,744,101; 5,744,305; and 5,489,678 as well as in “DNA ArraysMethods and Protocols,” Methods in Molecular Biology, by McGall, G. H.and Fidanza, J. A., and edited by Rampal J. B., Vol. 170, pp. 71-101,Humana Press, Inc., 2001, New York, N.Y., and in “Protection ofNucleosides for Oligonucleotide Synthesis,” Current Protocols in NucleicAcid Chemistry, ed. by Boyle, A. L., John Wiley & Sons, Inc., 2000, NewYork, N.Y., all of which are incorporated herein by reference in theirentirety. Preferably, the photolabile hydroxy protecting group isselected from the group consisting ofα-methyl-6-nitropiperonyloxycarbonyl (i.e, MeNPOC);o-nitrophenylethoxycarbonyl; o-nitrophenylethoxysulfonyl;3′,5′-dimethoxybenzoinoxycarbonyl and derivatives thereof, including butnot limited to 2-(2-nitrophenyl)-2-methylethoxycarbonyl and2-(2-nitro-6-chlorophenyl)-2-methylethylsulfonyl. As used herein theterm “derivatives thereof” refers to compounds that have one or moresubstituents on the aromatic (e.g., phenyl) ring or the alkylene chainportion of the photolabile hydroxy protecting group. For example, thephenyl ring moiety can further comprise one or more substituents such asalkoxy, alkyl, halide, cyano, amino, hydroxy, and nitro; and thealkylene chain portion can be substituted with alkyl, optionallysubstituted aryl, halide and alkoxy.

Preferably, R¹ and R² together form an amine protecting group. Morepreferably, R¹ and R² together form an amine protecting group of theformula: ═CH—N(CH₃)₂.

Another aspect of the present invention provides a process for producinga nucleoside. In particular, processes of the present invention utilizea halogenated nucleoside base to produce a non-halogenated nucleosidebase containing nucleoside. In particular, the present inventors havefound that the presence of a halogen on the nucleoside base allows theresulting nucleoside to be purified by methods other thanchromatography, preferably by recrystallization or precipitation,thereby allowing a large scale synthesis of a variety of nucleosides ornucleotides. In addition, purification by recrystallization orprecipitation significantly reduces the cost of producing a variety ofnucleosides.

The process of the present invention includes contacting a halogenatednucleoside base with an activated sugar under conditions sufficient toproduce a halogenated nucleoside base containing nucleoside. Thehalogenated nucleoside base is then reduced under conditions sufficientto produce a non-halogenated nucleoside base containing nucleoside.Preferably, the halogenated nucleoside base portion of the nucleoside isreduced by hydrogenation in the presence of a hydrogenation catalyst.

The processes of the present invention result in at least about 50%yield of the non-halogenated nucleoside base containing nucleoside fromthe halogenated nucleoside base. Preferably, the yield is at least about70%, and more preferably at least about 87%.

The non-halogenated nucleoside base containing nucleoside can be furtherbe converted to a phosphoramidite nucleoside, i.e., a nucleosidecomprising a phosphoramidite functional group attached to the sugarportion of the nucleoside. The phosphoramidite nucleosides are usefulintermediates in a synthesis of a variety of oligonucleosides andoligonucleotides.

In one aspect, the present invention provides a process for producing anucleoside comprising a hydropyrazolopyrimidine nucleoside base. Theprocess comprises hydrolyzing and reducing or reducing and hydrolyzingan iodopyrazolopyrimidine nucleoside of the formula:

under conditions sufficient to produce a hydropyrazolopyrimidinenucleoside of the formula:

wherein

-   -   R¹ is selected from the group consisting of hydrogen and alkyl;    -   R² is selected from the group consisting of hydrogen, alkyl, and        an amine protecting group, or R¹ and R² together form an amine        protecting group;    -   R³ is selected from the group consisting of alkyl, and a hydroxy        protecting group; and    -   each of Y¹, Y², Y³, Y⁴, Y⁵, and Y⁶ is independently selected        from the group consisting of hydrogen, halide, alkyl, —OR⁴,        wherein each R⁴ is independently selected from the group        consisting of hydrogen, alkyl, and a hydroxy protecting group or        two R⁴ groups form a diol protecting group, e.g., acetonide, or        Y² and Y⁴ together with the carbon atoms to which they are        attached and the carbon atom bearing Y³ and Y⁶ substituents        (i.e., C-3 carbon atom of the carbohydrate ring) form a five-to        seven membered ring. Such compounds are sometimes referred to as        “locked” nucleic acids, and examples of preparing locked nucleic        acids are illustrated by Singh et al., Chem. Comm., 1998,        455-456; and Wengel, Acc. Chem. Res., 1998, 32, 301-310, which        are incorporated herein by reference in their entirety.

When R⁴ is a hydroxy protecting group, preferably it is selected fromthe group consisting of an acid labile hydroxy protecting group and aphotolabile hydroxy protecting group. More preferably R⁴ is selectedfrom the group consisting of dimethoxytrityl, trityl, pixyl,1,1-bis(4-methoxyphenyl)-1-pyrenylmethyl (i.e., BMPM),α-methyl-6-nitropiperonyloxycarbonyl,2-(2-nitrophenyl)-2-methylethoxycarbonyl,2-(2-nitro-6-chlorophenyl)-2-methylethylsulfonyl and3′,5′-dimethoxybezoinoxycarbonyl.

The hydrolysis reaction replaces the alkoxide group (—OR³) with ahydroxy group (OH). Preferably, hydrolysis is conducted in an aqueoussolution in the presence of a base. Suitable bases include hydroxides,such as sodium hydroxide, lithium hydroxide, potassium hydroxide,calcium hydroxide, magnesium hydroxide; and the like. While a hydroxideis a preferred base, it should be appreciated that any base which hassufficient pKa to generate a hydroxide moiety from water can also beused. Alternatively, the hydrolysis reaction can also be conducted usingan acid catalyst, e.g., 2M aqueous HCl at 80° C., see for example, J.Chem. Soc. Chem. Comm., 1990, 19, 1380-1382.

The hydrolysis reaction temperature depends on a variety of factorsincluding concentration of each reagent, reaction medium, reaction time,etc. Typically, however, the hydrolysis reaction temperature is in therange of from about 60° C. to about 105° C. Preferably, the hydrolysisis conducted at a reaction temperature of from about 70° C. to about100° C., more preferably from about 80° C. to about 95° C., and mostpreferably from about 85° C. to about 90° C.

The reaction time depends on a variety of factors including theconcentration of each reagent, reaction temperature, and the base used.Typically, the hydrolysis reaction time in an aqueous solvent in thepresence of a hydroxide ranges from about 2 hrs to about 24 hrs,preferably from about 4 hrs to about 12 hrs, and more preferably fromabout 5 hrs to about 7 hrs. Generally, one skilled in the art willreadily recognize that the progress of reaction can be monitored byconventional analytical methods, such as TLC, HPLC and GC.

Hydrogenolysis, e.g., replacement of iodide with hydrogen, can beachieved by any conventional halogen reduction method known to one ofordinary skill in the art. Typically, the iodide is reduced byhydrogenation in the presence of a hydrogenation catalyst. Exemplaryhydrogenation catalysts that are useful in reducing iodide to hydrogeninclude, but are not limited to, palladium, platinum, rhodium, RaneyNickel®, and the like. In one particular embodiment of the presentinvention, palladium on carbon is used as the hydrogenation catalyst.Preferably, the hydrogenation reaction is conducted near the atmosphericpressure of hydrogen.

Suitable solvents for hydrogenation reaction include, but are notlimited to, alcohol, such as methanol, ethanol, isopropanol, and thelike; water; DMF; THF; methoxyethanol; and mixtures thereof. Typicallywater or a solvent mixture comprising water is used as the hydrogenationsolvent because the hydrogenation reaction product is soluble in water.Water is also preferred since it is non-flammable and is not subject tothe hazards in the presence of active hydrogenolysis catalysts.

The hydrolysis and reduction can be conducted in any sequence. While theproduct of the first step can be purified prior to conducting asubsequent reaction, it has been found by the present inventors thatsuch purification is not necessary. Typically, theiodopyrazolopyrimidine nucleoside of Formula I is hydrolyzed and theresulting product is subjected to a hydrogenation reaction without anypurification. The resulting hydropyrazolopyrimidine nucleoside ofFormula II can be conveniently purified by crystallization. Preferably,the yield of hydropyrazolopyrimidine nucleoside of Formula II from theiodopyrazolopyrimidine nucleoside of Formula I using the process of thepresent invention is at least about 50%, more preferably at least about70%, and most preferably at least about 90%.

The hydropyrazolopyrimidine nucleoside of Formula II can be used as anintermediate in synthesis of a variety of nucleosides including PPGphosphoramidite.

Thus, in another aspect of the present invention provide a process forproducing a PPG phosphoramidite of the formula:

from the hydropyrazolopyrimidine nucleoside of the formula:

wherein

-   -   R¹ is hydrogen and R² is an amine protecting group or R¹ and R²        together form an amine protecting group; and    -   one of R⁹ and R¹⁰ is a phosphoramidite and the other is a        hydroxy protecting group.

The PPG phosphoramidite producing step comprises:

-   -   (a) (i) contacting the hydropyrazolopyrimidine nucleoside of        Formula II with an amine protecting reagent under conditions        sufficient to produce an amine-protected nucleoside of the        formula:    -   (ii) contacting the amine-protected nucleoside of Formula V with        a hydroxy protecting reagent under conditions sufficient to        produce an amine/monohydroxy protected nucleoside of the        formula:

VI or

-   -   (i) contacting the hydropyrazolopyrimidine of Formula IV with a        hydroxy protecting reagent under conditions sufficient to        produce a monohydroxy protected nucleoside of the formula:    -   (ii) contacting the monohydroxy protected nucleoside of Formula        VII with an amine protecting reagent under conditions sufficient        to produce the amine/monohydroxy protected nucleoside of Formula        VI,        wherein    -   R¹ is hydrogen and R² is an amine protecting group or R¹ and R²        together form an amine protecting group; and    -   one of R⁷ and R⁸ is hydrogen and the other is a hydroxy        protecting group; and    -   (b) contacting the anine/monohydroxy protected nucleoside of        Formula VI with an activated phosphoramidite under conditions        sufficient to produce the PPG phosphoramidite of Formula III.

Any of the conventional amine protecting reagents can be used, such asthose disclosed in the above incorporated Beaucage and Iyer,Tetrahedron, 1992, 48, 2223-2311; T. W. Greene and P. G. M. Wuts,Protective Groups in Organic Synthesis, 3^(rd) edition, John Wiley &Sons, New York, 1999; and Harrison and Harrison et al., Compendium ofSynthetic Organic Methods, Vols. 1-8 (John Wiley and Sons, 1971-1996).In one particular embodiment, the amine protecting reagent is selectedfrom the group consisting of N,N-dialkyl formamide dialkylacetal, suchas N,N-dimethylformamide dimethylacetal; and N,N-dialkylacetamidedialkylacetal, such as N,N-dimethylacetamide dimethylacetal. Reactionconditions for protecting an amino group are well known to one skilledin the art.

Similarly, any of the conventional hydroky protecting groups can be usedin the processes of the present invention, such as those disclosed inthe above described Protective Groups in Organic Synthesis, 3^(rd)edition. In one particular embodiment, the hydroxy protecting reagent isan acid labile hydroxy protecting reagent, i.e., hydroxy protectinggroup which can be deprotected (i.e., removed) using an acid.Preferably, the acid labile hydroxy protecting reagent is selected fromthe group consisting of pixyl halide, such as pixylchloride; tritylhalide; monomethoxytrityl halide; dimethoxytrityl halide; and1,1-bis(4-methoxyphenyl)-1-pyrenylmethyl halide (i.e., BMPM halide).Reaction conditions for protecting a hydroxy group are also well knownto one skilled in the art and are also disclosed in the aboveincorporated Beaucage and Iyer, Tetrahedron, 1992, 48, 2223-2311; T. W.Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 3^(rd)edition, John Wiley & Sons, New York, 1999; Boyle, Ann L. (Editor),Current Protocols in Nucleic Acid Chemistry, John Wiley and Sons, NewYork, 2000; and Harrison and Harrison et al., Compendium of SyntheticOrganic Methods, Vols. 1-8 (John Wiley and Sons, 1971-1996).

In another embodiment, the hydroxy protecting reagent is a photolabilehydroxy protecting reagent. Suitable photolabile hydroxy protectingreagents are well known to one skilled in the art and include thosedisclosed in the above incorporated U.S. Pat. Nos. 5,889,165; 5,744,101;5,744,305; and 5,489,678; “DNA Arrays Methods and Protocols,” Methods inMolecular Biology, by McGall, G. H. and Fidanza, J. A., and edited byRampal J. B., Vol. 170, pp. 71-101, Humana Press, Inc., 2001, New York,N.Y.; and “Protection of Nucleosides for Oligonucleotide Synthesis,”Current Protocols in Nucleic Acid Chemistry, ed. by Boyle, A. L., JohnWiley & Sons, Inc., 2000, New York, N.Y. Preferably, the photolabilehydroxy protecting reagent is selected from the group consisting of1-(3,4-methylenedioxy-6-nitrophenyl)ethyl chloroformate,2-(2-nitrophenyl)-2-methylethyl chloroformate,2-(2-nitro-6-chlorophenyl)-2-methylethylsulfonyl chloride, and3′,5′-dimethoxybezoinoxyl chloroformate.

Nucleosides of the present invention comprising ahydropyrazolopyrimidine nucleoside base and a photolabile hydroxyprotecting group on the sugar moiety are useful in a variety ofapplications. In particular, these nucleosides are useful insynthesizing a library of oligonucleotides (i.e., array ofoligonucleotides) using conventionally well known solid phaseoligonucleotide array synthesis such as those disclosed in the aboveincorporated U.S. Pat. Nos. 5,889,165; 5,744,101; 5,744,305; and5,489,678; and “DNA Arrays Methods and Protocols,” Methods in MolecularBiology, by McGall, G. H. and Fidanza, J. A., and edited by Rampal J.B., Vol. 170, pp. 71-101, Humana Press, Inc., 2001, New York, N.Y.

In one aspect of the present invention, the activated phosphoramidite isof the formula:

where X² is a leaving group, preferably halide or diisopropylamino,i.e., a moiety of the formula —N(i-Pr)₂. The reaction between theactivated phosphoramidite of Formula VIII and the amine/monohydroxyprotected nucleoside of Formula VI is typically carried out at atemperature range of from about 0° C. to about 40° C., preferably fromabout 10° C. to about 35° C., and more preferably from about 20° C. toabout 30° C. Suitable reaction solvents are inert organic solvents,including halogenated organic solvents, such as methylene chloride andchloroform; ethers, such as tetrahydrofuran and diethyl ether; aromatichydrocarbons, such as toluene, xylenes and halogenated phenyls;hydrocarbons; and other organic solvents which are substantially inertto the reaction conditions and in which the reagents are soluble. Itshould be appreciated that when organic solvents which have a higherboiling point is used, the reaction temperature range cancorrespondingly be extended.

The processes of the present invention can also include producing thenucleoside of Formula I, which comprises contacting aniodopyrazolopyrimidine of the formula:

with an activated sugar of the formula:

under conditions sufficient to produce the nucleoside of Formula I,where R¹, R², R³, Y¹, Y², Y³, Y⁴, Y⁵, and Y⁶ are those defined above;and X¹ is a leaving group. Preferably, X¹ is a halide, and morepreferably chloride.

Generally, the process for producing the nucleoside of Formula Iinvolves contacting the iodopyrazolopyrimidine of Formula IX with a basehaving a sufficient pKa to deprotonate the pyrazolyl nitrogen atom andcontacting the deprotonated iodopyrazolopyrimidine with the activatedsugar of Formula X. Such deprotonation of the iodopyrazolopyrimidine ofFormula IX by the base can occur in situ, as described in detail below.The reaction temperature for producing the nucleoside of Formula I istypically in the range of from about 5° C. to about 60° C., preferablyfrom about 10° C. to about 40° C., and more preferably from about 20° C.to about 30° C. Suitable reaction solvents include, but are not limitedto, alcohols, such as methanol, ethanol, isopropanol, and the like;relatively polar organic solvents, such as DMF and acetonitrile; water;and mixtures thereof.

Alternatively, the iodopyrazolopyrimidine of Formula IX and theactivated sugar of Formula X can be combined in a reaction vessel in thepresence of a base. In this alternative embodiment, the reactionconditions are selected such that the reaction rate between theiodopyrazolopyrimidine of Formula IX and the activated sugar of FormulaX is significantly higher than the reaction rate between the base andthe activated sugar of Formula X.

Useful bases for producing the nucleoside of Formula I from theiodopyrazolopyrimidine of Formula IX and the activated sugar of FormulaX include, but are not limited to, bases in which the correspondingacids have pKa of at least about 9, preferably at least about 10, andmore preferably at least about 12. Exemplary bases include, but are notlimited to, alkoxides, such as alkaline or alkaline-earth metalmethoxides, ethoxides, propoxides, isopropoxides, butoxides,tert-butoxides, and the like; hydrides, such as sodium hydride, calciumhydride, potassium hydride, and the like; and organic bases, such asamines, e.g., tertiary amines, DBU and DBN.

The processes of the present invention provide the nucleoside of FormulaI from the iodopyrazolopyrimidine of Formula IX and the activated sugarof Formula X at a yield of at least about 35%. Preferably, the yield isat least about 50%, and more preferably at least about 65%.

The processes of the present invention can also include producing theiodopyrazolopyrimidine of Formula IX (e.g., where R¹ and R² arehydrogen) comprising:

-   -   (i) contacting a pyrimidinone of the formula:        with a halogenating agent and a formylating agent under        conditions sufficient to produce a dihalopyrimidine        carboxyaldehyde of the formula:        where each X³ is independently selected from the group        consisting of F, Cl, Br and I;    -   (ii) contacting the dihalopyrimidine carboxyaldehyde of Formula        XII with hydrazine under conditions sufficient to produce a        halopyrazolopyrimidine of the formula:    -   (iii) contacting the halopyrazolopyrimidine of Formula XIII with        an alkoxide of the formula R³—OM, where R³ is alkyl and M is a        metal, to produce an alkoxypyrazolopyrimidine of the formula:        and    -   (iv) iodinating the alkoxypyrazolopyrimidine of Formula XIV with        an iodinating agent under conditions sufficient to produce the        iodopyrazolopyrimidine of Formula IX.

Preferably, the halogenating agent is selected from the group consistingof POCl₃, iodine monochloride (i.e., ICl), N-iodosuccinamide and SOCl₂.The formylation and halogenation reactions can be conveniently conductedin a single reaction condition. For example, when POCl₃ is used as thehalogenating agent, dimethyl formamide (DMF) can be used as a reactionsolvent and a formylating agent to provide the dihalopyrimidinecarboxyaldehyde of Formula XII directly from the pyrimidinone of FormulaXI. In this embodiment, the reaction mixture is typically combined atabout 0° C. The resulting mixture then is heated to a temperature rangeof from about 40° C. to about 100° C., preferably from about 60° C. toabout 100° C., and more preferably from about 90° C. to about 98° C. Theresulting dihalopyrimidine carboxyaldehyde of Formula XII precipitatesout of an aqueous solution and can be further purified byrecrystallization. The yield of combined formylation and halogenationreaction is typically at least about 50%, preferably at least about 60%,and more preferably at least about 70%. Other suitable Vilsmeierformylation/halogenating reagent combinations include any compoundhaving a formyl group attached to a secondary amino group in combinationwith a halogenating agent. Exemplary formylating reagents include1-formylpiperidine, 1-formylmorpholine and triformamide. Exemplaryhalogenating agents include oxalyl chloride, PCl₅, Br₂/PPh₃ and SOX₂,where X is Cl or Br.

Reacting the dihalopyrimidine carboxyaldehyde of Formula XII withhydrazine then affords the halopyrazolopyrimidine of Formula XIII.Alternatively, hydrazine monohydrate can be used rather than anhydroushydrazine. Typically, this reaction is conducted in the presence of abase, preferably a trialkyl amine base, such as triethylamine. Theresulting halopyrazolopyrimidine of Formula XIII can be purified byrecrystallization.

The halopyrazolopyrimidine of Formula XIII is then reacted with analkoxide of the formula R³—OM, where R³ is alkyl and M is a metal, todisplace the halide (X³), thereby producing the alkoxypyrazolopyrimidineof Formula XIV. It should be appreciated that while the empiricalformula of the alkoxide is written as R³—OM, depending on the nature ofthe metal, there can be more than one alkoxide group per metal. Forexample, when the metal is a divalent metal, e.g., alkaline earth metalor a transition metal such as Mg or Ca, the valence of these metals are+2; therefore, there are two alkoxide groups associated with each metalin the empirical formula. Displacement of the halide (X³) by thealkoxide is conveniently carried out in an alcoholic solvent, preferablythe alcohol corresponding to the alkoxide. Generally, the reaction isheated, preferably to the solvent's refluxing temperature, to facilitatethe reaction. The resulting mixture is then cooled, neutralized byadding an acid, and precipitated to afford the alkoxypyrazolopyrimidineof Formula XIV.

Reaction of the alkoxypyrazolopyrimidine of Formula XIV with ahalogenating agent provides a halogenated nucleoside base. The presentinventors have found that the presence of a halogen on the nucleosidebase allows purification of the resulting halogenated nucleoside basecontaining nucleoside by recrystallization; thus, providing a means fora cost effective large scale nucleoside synthesis. The halogen can bechlorine, bromine or iodine; however, iodine is preferred as it providesthe highest mass. Without being bound by any theory, it is generallybelieved that high mass is one of the factors that contribute in thecompound being a solid, which allows the compound to be purified byrecrystallization or precipitation.

Iodination of the alkoxypyrazolopyrimidine of Formula XIV can beaffected by any electrophilic iodinating agent which is compatible withfunctional groups that are present in the alkoxypyrazolopyrimidine ofFormula XIV. Exemplary iodinating agents include iodine monochloride,N-iodosuccinimide and other iodinating agents known to one skilled inthe art. Preferably, the iodinating agent is selected from the groupconsisting of iodine monochloride and N-iodosuccinimide.

In one particular embodiment, the iodopyrazolopyrimidine of Formula IXis produced by reacting the alkoxypyrazolopyrimidine of Formula XIV withiodine monochloride. Typically, the iodination is conducted in anaqueous solution under refluxing conditions. The reaction can alsoinclude adding a mild base to neutralize or buffer the effect of acidthat is generated in the reaction mixture. Suitable mild bases includecarboxylates, such as metal acetates; and other salts of acids having apKa of from about 4 to about 6. After the reaction, any remaining iodidemonochloride is destroyed (i.e., neutralized, removed or converted toother compound) by adding a reducing agent such as sodium metabisulfiteor other suitable reducing agents. Preferably, the reaction solvent isselected such that the resulting iodopyrazolopyrimidine of Formula IXprecipitates out of the reaction mixture, which can be further purifiedby dissolving in hot DMF and precipitating it by addition of water.

Additional objects, advantages, and novel features of this inventionwill become apparent to those skilled in the art upon examination of thefollowing examples thereof, which are not intended to be limiting.

EXAMPLES Example 1

This example illustrate a method for producing2-amino-4,6-dichloropyrimidine-5-carboxaldehyde (2).

Absolute DMF (210 ml, 1.38 mol) was added drop wise to an ice-coldsolution of POCl₃ (900 ml) over a 20 min period. The ice-bath wasremoved and 150 g (1.17 mol) of powdered2-amino-6-hydroxypyrimidine-4(3H)-one (1) was added over a 20 minperiod. The mixture was then heated to 100° C. and stirred for 3-4 h.The solution was cooled to room temperature and poured into 10 L of icewater (4 L of crushed ice diluted to 10 L with water). This aqueoussolution was then heated to 50° C. and stirred for 2 h. The mixture wasrefrigerated overnight and the precipitate was filtered and dried toprovide 160 g (71% yield) of2-amino-4,6-dichloropyrimidine-5-carboxaldehyde (2).

Example 2

This example illustrate a method for producing4-chloro-6-aminopyrazolo[3,4-d]pyrimidine (3).

To a mixture of 2-amino-4,6-dichloropyrimidine-5-carboxaldehyde (2) (150g, 0.785 mol), THF (2.7 L) and triethylamine (125 ml) was addedanhydrous hydrazine (25.5 ml in 600 ml of water) drop wise over 25 min.Alternatively, hydrazine monohydrate can be used instead of anhydroushydrazine. The mixture was stirred for 1 h. The solids were filtered offand the filtrate was evaporated to remove about 80% of the THF. Waterwas added and the resulting precipitate was filtered and dried undervacuum. The solid was then dissolved in a minimum volume of hot DMF andprecipitate by addition of water to provide 112 g (85% yield) of4-chloro-6-aminopyrazolo[3,4-d]pyrimidine (3).

Example 3

This example illustrates a method for producing4-methoxy-6-aminopyrazolo[3,4-d]pyrimidine (4).

Compound 3 (70.4 g, 0.416 mol) was refluxed in methanolic sodiummethoxide solution (25 g sodium dissolved in 1 L methanol) for 2 h. Thereaction mixture was cooled to room temperature and neutralized byaddition of acetic acid (75 ml). The mixture was evaporated to drynessand the solid was triturated in 600 ml of water, filtered and dried toprovide 67.5 g (99% yield) of 4-methoxy-6-aminopyrazolo[3,4-d]pyrimidine(4).

Example 4

This example illustrates a method for producing3-iodo-4-methoxy-6-aminopyrazolo[3,4-d]pyrimidine (5).

A mixture of compound 4 (105 g, 0.636 mol), sodium acetate (263 g) andiodine monochloride (140 g) was mechanically stirred in 1.3 L of waterat a temperature between 92-98° C. for 5 h. The mixture was cooled toroom temperature and a solution of sodium metabisulfite (139 g in 500 mlof water) was added. The mixture was stirred for 10 minutes and thenfiltered. The solid was rinsed with water and dried. The resulting driedsolid was dissolved in 840 ml of hot DMF and precipitate by addition of800 ml of water. The mixture was cooled in an ice-bath and the solid wasfiltered and dried to provide 144 g (78% yield) of3-iodo-4-methoxy-6-aminopyrazolo[3,4-d]pyrimidine (5).

Example 5

This example illustrates a method for producing3-iodo-4-methoxy-6-amino-1-(2-deoxy-3,5-di-O-toluoyl-β-D-erythro-pentofuranosyl)1H-pyrazolo[3,4-d]pyrimidine (6).

To a suspension of Compound 5 (67 g, 0.230 mol) in 650 ml of methanolwas added a solution of 15.22 g KOH (15% water by weight) dissolved in100 ml of methanol. The mixture was stirred for 5 min and thenevaporated to dryness. The solid was dissolved in 270 ml of anhydrousDMF and then diluted with 1.55 L of anhydrous acetonitrile (i.e., MeCN).The chlorosugar derivative A (107 g) was added immediately after theMeCN dilution. The mixture was stirred vigorously for 2.5 hours and thecrude product was filtered. The damp solid was triturated in 350 ml ofwater, filtered and dried to provide 97.7 g (66% yield) of3-iodo-4-methoxy-6-amino-1-(2-deoxy-3,5-di-O-toluoyl-β-D-erythro-pentofuranosyl)1H-pyrazolo[3,4-d]pyrimidine (6).

Example 6

This example illustrates a method for producing3-iodo-4-methoxy-6-amino-1-(2′-deoxy-β-D-erythro-pentofuranosyl)pyrazolo[5,4-d]pyrimidine(7).

Compound 6 (219 g, 0.341 mol) was suspended in 970 ml of methanol. Asolution of 1 M NaOMe in methanol (130 ml) was added and the mixture wasrefluxed for 1 h. The flask was placed in the freezer for 5 hours andthe crystals were filtered and rinsed sparingly with cold methanol toprovide 89.6 g (65% yield) of3-iodo-4-methoxy-6-amino-1-(2′-deoxy-β-D-erythro-pentofuranosyl)pyrazolo[5,4-d]pyrimidine(7).

The yield can be increased to 71% by cooling the reaction mixture in thefreezer overnight.

Example 7

This example illustrates a method for producing1-(2′-deoxy-β-D-erythro-pentofuranosyl)-6-amino-5-hydropyrazolo[5,4-d]pyrimidine-4-one(8).

Compound 7 (73.35 g, 0.180 mol) was stirred in a solution of 21 g NaOHin 885 ml water at 90° C. for 6 h. The clear solution was cooled to roomtemperature and then transferred to a hydrogenation bottle containing3.6 g of 5% Pd/C (50% water content by weight, Aldrich Chemical Co.,Milwaukee, Wis.). The mixture was shaken under 40 psi of hydrogen for 90min. The mixture was filtered through Celite® and the filtrate wasneutralized to pH 7 using concentrated HCl, followed by a fine pHadjustment with acetic acid. The solution was placed in a refrigeratorovernight and the crystals that formed were filtered, rinsed sparinglywith ice-cold water, and dried to provide 41.5 g (87% yield) of1-(2′-deoxy-β-D-erythro-pentofuranosyl)-6-amino-5-hydropyrazolo[5,4-d]pyrimidine-4-one(8).

Example 8

This example illustrates a method for producing1-(2′-deoxy-β-D-erythro-pentofuranosyl)-6-formadino-5-hydropyrazolo[5,4-d]pyrimidine-4-one(9).

Compound 8 (101 g, 0.378 mol) was dissolved in 325 ml of dry DMF.N,N-Dimethylformamide dimethylacetal (150 ml) was added. The mixture wasstirred for 4 hours. The solvents were evaporated off and the residuewas twice evaporated from xylenes. The foam was then triturated in 200ml of absolute ethanol. The mixture was diluted by addition of 1.6 L ofether and the solid was filtered and dried to provide 119 g (97% yield)of1-(2′-deoxy-β-D-erythro-pentofuranosyl)-6-formadino-5-hydropyrazolo[5,4-d]pyrimidine-4-one(9).

Example 9

This example illustrates a method for producing an acid labile DMTrhydroxy protecting group derivative of Compound 9.

Dimethoxytrityl chloride (8.0 g) was added to a solution of Compound 9(6.42 g, 19.92 mmol) in 120 ml dry pyridine. The reaction solution wasstirred for 3 h at room temperature. The reaction was monitored by TLC(5% methanol in ethyl acetate) and add more DMTrCl as necessary. Thecompleted reaction solution was poured into 600 ml of 5% sodiumbicarbonate. The mixture was extracted with 600 ml of ethyl acetate andthe extract was dried over sodium sulfate and evaporated. The residuewas purified using silica gel chromatography eluting with 5% methanol inethyl acetate. The product fractions were evaporated affording 10.6 g(85% yield) of Compound 10 as a foam.

Example 10

This example illustrates a method for producing a photolabile MeNPOChydroxy protecting group derivative of Compound 9.

Compound 11 is synthesized by a modified procedure described by McGall,G. H. and Fidanza, J. A., “DNA Arrays Methods and Protocols,” Edited byRampal J. B., Methods in Molecular Biology, 170: 71-101 (2001), HumanaPress, Inc., New York, N.Y., in particular sections 3.1.2 and 3.1.3.

Example 11

This example illustrates a method for producing PPG phosphoramidite (12)comprising an acid labile hydroxy protecting group DMTr.

To a solution of Compound 10 (9.15 g, 14.65 mmol) dissolved in 356 ml ofdry methylene chloride, containing 7.6 ml of N,N-diisopropylethylamine,was added 5.5 ml of 2-cyanoethyl diisopropylchlorophosphoramidite. Thesolution was stirred under argon for 2 hrs. at room temperature. Thesolution was treated with 10 ml of methanol and then poured into 400 mlof 5% sodium bicarbonate solution. The mixture was extracted with ethylacetate (500 ml) and the extract was dried over sodium sulfate, filteredand evaporated. The residue was purified by silica gel chromatographyeluting with 3% methanol in ethyl acetate (2% triethylamine). The pureproduct fractions were evaporated. The residue was dissolved in aminimum volume of ether, which was added drop wise to filtered hexanes.The precipitate was filtered and dried to provide 9.8 g (81% yield) ofPPG phosphoramidite (12).

Example 12

This example illustrates a method for producing PPG phosphoramidite (13)comprising a photolabile hydroxy protecting group MeNPOC.

Compound 13 is synthesized by a modified procedure described by McGall,G. H. and Fidanza, J. A., “DNA Arrays Methods and Protocols,” Edited byRampal J. B., Methods in Molecular Biology, 170: 71-101 (2001), HumanaPress, Inc., New York, N.Y., in particular section 3.1.6.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1. A PPG phosphoramidite comprising a photolabile hydroxy protectinggroup, wherein said phosphoramidite nucleoside is of the formula:

wherein R¹ is selected from the group consisting of hydrogen and alkyl;R² is selected from the group consisting of hydrogen, alkyl, and anamine protecting group, or R¹ and R² together from an amine protectinggroup; each of Z¹, Z², Z⁴, and Z⁶ is independently selected from thegroup consisting of hydrogen halide, alkyl, —OR¹¹, wherein each R¹¹ isindependently selected from the group consisting of hydrogen, alkyl, anda hydroxy protecting group or two R¹¹ groups form a diol protectinggroup, or Z² and Z⁴ together with the carbon atoms to which they areattached and C-3 carbon atom of the carbohydrate ring form a five-toseven membered ring; and one of Z³ or Z⁵ is —OR¹² and the other is—OR¹³, where R¹² is a photolabile hydroxy protecting group and R¹³ is aphosphoramidite.
 2. The PPG phosphoramidite according to claim 1 of theformula:

wherein R¹, R², Z³ and Z⁵ are those defined in claim
 1. 3. The PPGphosphoramidite according to claim 2, wherein Z³ is —OR¹³ and Z⁵ is—OR¹², where R¹² and R¹³ are those defined in claim
 1. 4. The PPGphosphoramidite according to claim 3, wherein the photolabile hydroxyprotecting group is selected from the group consisting ofα-methyl-6-nitropiperonyloxycarbonyl,2-(2-nitrophenyl)-2-methylethoxycarbonyl,2-(2-nitro-6-chlorophenyl)-2-methylethylsulfonyl, and3′,5′-dimethoxybezoinoxycarbonyl.
 5. The PPG phosphoramidite accordingto claim 4, wherein R¹ and R² together form an amine protecting group.6. The PPG phosphoramidite according to claim 5, wherein R¹ and R²together form an amine protecting group of the formula: ═CH—N(CH₃)₂. 7.A process for producing a non-halogenated nucleoside base containingnucleoside comprising: (a) contacting a halogenated nucleoside base withan activated sugar under conditions sufficient to produce a halogenatednucleoside base containing nucleoside; and (b) reducing said halogenatednucleoside base containing nucleoside under conditions sufficient toproduce said non-halogenated nucleoside base containing nucleoside. 8.The process of claim 7, wherein said non-halogenated nucleoside basecontaining nucleoside is purified by recrystallization.
 9. The processof claim 7, wherein the yield of said non-halogenated nucleoside basecontaining nucleoside from said halogenated nucleoside base is at leastabout 50%.
 10. The process of claim 7, wherein said halogenatednucleoside base containing nucleoside reducing step compriseshydrogenation of said halogenated nucleoside base containing nucleosidein the presence of a hydrogenation catalyst.
 11. The process of claim 7,wherein said non-halogenated nucleoside base containing nucleoside isused in a synthesis of a phosphoramidite nucleoside.
 12. The process ofclaim 11, wherein said phosphoramidite nucleoside is used in a synthesisof an oligonucleotide or an oligonucleotide.
 13. A process for producinga nucleoside comprising a hydropyrazolopyrimidine nucleoside base, saidprocess comprising hydrolyzing and reducing or reducing and hydrolyzingan iodopyrazolopyrimidine nucleoside of the formula:

under conditions sufficient to produce a hydropyrazolopyrimidinenucleoside of the formula:

wherein R¹ is selected from the group consisting of hydrogen and alkyl;R² is selected from the group consisting of hydrogen, alkyl, and anamine protecting group, or R¹ and R² together form an amine protectinggroup; R³ is selected from the group consisting of alkyl, and a hydroxyprotecting group; and each of Y¹, Y², Y³, Y⁴, Y⁵, and Y⁶ isindependently selected from the group consisting of hydrogen, halide,alkyl, —OR⁴, wherein each R⁴ is independently selected from the groupconsisting of hydrogen, alkyl, and a hydroxy protecting group or two R⁴groups form a diol protecting group, or Y² and Y⁴ together with thecarbon atoms to which they are attached to and C-3 carbon atom of thecarbohydrate ring form a five-to seven membered ring.
 14. The process ofclaim 13, wherein R¹, R², Y¹, Y², Y⁴, and Y⁶ are hydrogen, and Y³ and Y⁵are —OR⁴.
 15. The process of claim 14, wherein R⁴ are hydrogen.
 16. Theprocess of claim 15 further comprising producing a PPG phosphoramiditeof the formula:

from said hydropyrazolopyrimidine nucleoside, wherein R¹ is hydrogen andR² is an amine protecting group or R¹ and R² together form an amineprotecting group; and one of R⁹ and R¹⁰ is a phosphoramidite and theother is a hydroxy protecting group, said PPG phosphoramidite producingstep comprises: (a) (i) contacting said hydropyrazolopyrimidinenucleoside with an amine protecting reagent under conditions sufficientto produce an amine-protected nucleoside of the formula:

(ii) contacting said amine-protected nucleoside with a hydroxyprotecting reagent under conditions sufficient to produce anamine/monohydroxy protected nucleoside of the formula:

or (i) contacting said hydropyrazolopyrimidine with a hydroxy protectingreagent under conditions sufficient to produce a monohydroxy protectednucleoside of the formula:

(ii) contacting said monohydroxy protected nucleoside with an amineprotecting reagent under conditions sufficient to produce anamine/monohydroxy protected nucleoside of the formula:

wherein R¹ is hydrogen and R² is an amine protecting group or R¹ and R²together form an amine protecting group; and one of R⁷ and R⁸ ishydrogen and the other is a hydroxy protecting group; and (b) contactingsaid amine/monohydroxy protected nucleoside with an activatedphosphoramidite under conditions sufficient to produce said PPGphosphoramidite.
 17. The process of claim 16, wherein said amineprotecting reagent is selected from the group consisting ofN,N-dialkylformamide dialkylacetal, and N,N-dialkylacetamidedialkylacetal.
 18. The process of claim 16, wherein said hydroxyprotecting reagent is a photolabile hydroxy protecting reagent.
 19. Theprocess of claim 18, wherein said photolabile hydroxy protecting reagentis selected from the group consisting of1-(3,4-methylenedioxy-6-nitrophenyl)ethyl chloroformate,2-(2-nitrophenyl)-2-methylethyl chloroformate,2-(2-nitro-6-chlorophenyl)-2-methylethylsulfonyl chloride and3′,5′-dimethoxybezoinoxyl chloroformate.
 20. The process of claim 16,wherein said hydroxy protecting reagent is an acid labile hydroxyprotecting reagent.
 21. The process of claim 20, wherein said acidlabile hydroxy protecting reagent is selected from the group consistingof trityl halide, monomethoxytrityl halide and dimethoxytrityl halide.22. The process of claim 16, wherein said activated phosphoramidite isof the formula:

wherein X² is a leaving group.
 23. The process of claim 22, wherein x isselected from the group consisting of halide and diisopropylamino. 24.The process of claim 22, wherein R⁹ is dimethoxytrityl and R¹⁰ is aphosphoramidite moiety of the formula —P[N(i-Pr)₂]OCH₂CH₂CN.
 25. Theprocess of claim 13 further comprising producing said nucleoside ofFormula I, wherein said nucleoside of Formula I producing stepcomprises: contacting an iodopyrazolopyrimidine of the formula:

with an activated sugar of the formula:

under conditions sufficient to produce said nucleoside of Formula I,wherein R¹, R², R³, Y¹, Y², Y³, Y⁴, Y⁵, and Y⁶ are those defined claim13; and X¹ is a leaving group.
 26. The process of claim 25 furthercomprising producing said iodopyrazolopyrimidine nucleoside of Formula Ifrom a pyrimidinone of the formula:

said iodopyrazolopyrimidine nucleoside producing process comprising: (i)contacting said pyrimidinone with a halogenating agent and a formylatingagent under conditions sufficient to produce a dihalopyrimidinecarboxyaldehyde of the formula:

wherein each X³ is independently selected from the group consisting ofF, Cl, Br and I; (ii) contacting said dihalopyrimidine carboxyaldehydewith hydrazine under conditions sufficient to produce ahalopyrazolopyrimidine of the formula:

(iii) contacting said halopyrazolopyrimidine with an alkoxide of theformula R³—OM, wherein R³ is alkyl and M is a metal, to produce analkoxypyrazolopyrimidine of the formula:

and (iv) iodinating said alkoxypyrazolopyrimidine with an iodinatingagent under conditions sufficient to produce saidiodopyrazolopyrimidine.
 27. The process of claim 26, wherein saidhalogenating agent is selected from the group consisting of POCl₃,iodine monochloride, N-iodosuccinamide and SOCl₂.
 28. The process ofclaim 26, wherein said formylating agent is a compound comprising aformyl group attached to a secondary amino group.
 29. The process ofclaim 28, wherein said formylating agent is selected from the groupconsisting of dimethyl formamide, 1-formylpiperidine, 1-formylmorpholineand triformamide.
 30. The process of claim 26, wherein said iodinatingagent is selected from the group consisting of iodine monochloride andN-iodosuccinimide.
 31. A process for producing a nucleoside comprising:(a) contacting an iodopyrazolopyrimidine of the formula:

with an activated sugar of the formula:

under conditions sufficient to produce an deoxy iodopyrazolopyrimidinenucleoside of the formula:

(b) producing an amino dihydro hydropyrazolopyrimidine nucleoside fromsaid deoxy iodopyrazolopyrimidine nucleoside, wherein said amino dihydrohydropyrazolopyrimidine nucleoside is of the formula:

wherein R³ is alkyl; R⁵ and R⁶ are hydroxy protecting groups; and X¹ isa leaving group.
 32. The process of claim 31, wherein said step ofproducing said amino dihydro hydropyrazolopyrimidine nucleosidecomprises removing said hydroxy protecting groups R⁵ and R⁶; hydrolyzing—OR³ group; and reducing the iodine.
 33. The process of claim 31 furthercomprising: (c) contacting said amino dihydro hydropyrazolopyrimidinenucleoside with an amine protecting reagent under conditions sufficientto produce an amine protected nucleoside of the formula:

(d) contacting said amine protected nucleoside with a hydroxy protectingreagent under conditions sufficient to produce an amine/monohydroxyprotected nucleoside of the formula:

and (e) contacting said amine/monohydroxy protected nucleoside with anactivated phosphoramidite of the formula:

under conditions sufficient to produce a PPG phosphoramidite of theformula:

wherein R¹ is hydrogen; R² is an amine protecting group; or R¹ and R²together form an amine protecting group; R⁴ is a hydroxy protectinggroup; and X² is a leaving group.
 34. The process of claim 33, whereinX² is selected from the group consisting of halide, and —N(i-Pr)₂. 35.The process of claim 33, wherein R¹ and R² together form a nitrogenprotecting group of the formula: ═CH—N(CH₃)₂.
 36. The process of claim35, wherein R⁴ is selected from the group consisting of an acid labilehydroxy protecting group and a photolabile hydroxy protecting group. 37.The process of claim 36, wherein R⁴ is selected from the groupconsisting of dimethoxytrityl, trityl, pixyl,1,1-bis(4-methoxyphenyl)-1-pyrenylmethyl,α-methyl-6-nitropiperonyloxycarbonyl,2-(2-nitrophenyl)-2-methylethoxycarbonyl,2-(2-nitro-6-chlorophenyl)-2-methylethylsulfonyl and3′,5′-dimethoxybezoinoxycarbonyl.
 38. The process of claim 31, whereinsaid step (b) comprises reducing the iodide by hydrogenation.
 39. Theprocess of claim 31, wherein said iodopyrazolopyrimidine is producedfrom a pyrimidinone of the formula:

said iodopyrazolopyrimidine producing step comprising: (i) contactingsaid pyrimidinone with a halogenating agent and a formylating agentunder conditions sufficient to produce a dihalopyrimidinecarboxyaldehyde of the formula:

wherein each X³ is independently selected from the group consisting ofF, Cl, Br and I; (ii) contacting said dihalopyrimidine carboxyaldehydewith hydrazine under conditions sufficient to produce ahalopyrazolopyrimidine of the formula:

(iii) contacting said halopyrazolopyrimidine with an alcohol of theformula R³—OH to produce an alkoxypyrazolopyrimidine of the formula:

and (iv) iodinating said alkoxypyrazolopyrimidine with an iodinatingagent under conditions sufficient to produce saidiodopyrazolopyrimidine.
 40. The process of claim 39, wherein saidhalogenating agent is selected from the group consisting of POCl₃,iodine monochloride, N-iodosuccinamide and SOCl₂.
 41. The process ofclaim 40, wherein said halogenating agent is selected from the groupconsisting of POCl₃ and SOCl₂.
 42. The process of claim 39, wherein saidformylating agent is selected from the group consisting of dimethylformamide, 1-formylpiperidine, 1-formylmorpholine and triformamide. 43.The process of claim 39, wherein said iodinating agent is selected fromthe group consisting of iodine monochloride and N-iodosuccinimide.